Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of insulin signaling and a novel therapeutic target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. However, besides a role in insulin signaling, PTP1B is also implicated in several other physiological processes including leptin and integrin mediated pathways. In addition, the molecular basis for the observed tissue-specific effects on insulin action due to PTP1B deletion and the unexpected phenotype of resistance to diet-induced obesity displayed by the PTP1B knockout mice is not well understood. Because PTPIB may be a regulator of multiple signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance of PTPIB in these processes. This is an important prerequisite for the development of PTP 1B-based therapeutics for type 2 diabetes and obesity. The goals of this proposal are 1) to further define the functional role of PTP1B in cellular signaling using chemical genetics and interaction proteomic approaches, and 2) to develop novel activity-based PTP probes for global analysis of PTP activity in the whole proteome. Specifically, we will employ small molecule, potent and selective PTPIB inhibitors developed in our laboratory to delineate the physiological roles of PTP1B in insulin, leptin, and integrin signaling. We will apply a high-affinity PTPIB substrate-trapping mutant in combination with mass spectrometry for rapid isolation, identification, and characterization of physiological substrates of PTP1B. This will help elucidate the function of this enzyme as well as assignment of PTP1B to a specific signaling pathway. Finally, we will develop an activity-based proteomic technology that utilizes PTP specific probes to interrogate the function of the PTPs in the whole proteome both in normal physiology and in pathological conditions and to study compensatory changes in PTP activity in response to PTP1B deletion. We believe that the fusion of chemical genetics with proteomics will provide the most direct path to the molecular understanding of PTPIB in human physiology and pathogenesis.